The specific gravity of a test sample, such as urine or serum, is a measure of the relative proportions of solid material dissolved in the test sample to the total volume of the test sample. In general, the specific gravity of a test sample is a measure of the relative degree of concentration or the relative degree of dilution of the test sample. The specific gravity of many urine samples can be accurately correlated to the ionic strength, or ion concentration, of the test sample. Typically, as the number of urine components that are measured increases, the correlation of ionic strength to urine specific gravity also increases. With regard to urine samples, the assay for specific gravity helps interpret the results of the other assays performed in a routine urinalysis.
Clinically, under appropriate and standardized conditions of fluid restriction or increased fluid intake, the specific gravity of a urine sample measures the concentrating and diluting abilities of the kidneys of an individual. The specific gravity of urine ranges from about 1.005 to about 1.030, and usually is in the range from about 1.010 to about 1.025. A specific gravity of about 1.025 or above in a random first morning urine specimen indicates a normal concentrating ability of the kidneys.
Either an abnormally low or an abnormally high urine specific gravity is clinically significant. Therefore, accurate and reliable specific gravity assays of urine and other aqueous test samples must be available for both laboratory and home use. The assays must provide an accurate measurement of abnormally low and abnormally high specific gravities, such that a correct diagnosis can be made and correct medical treatment implemented, monitored and maintained.
For example, diabetes insipidus is characterized by excreting large urine volumes of low specific gravity, and is a severe example of impaired kidney concentrating ability. The urine specific gravity of individuals suffering from diabetes insipidus usually ranges between 1.001 and 1.003. Low urine specific gravity also occurs in persons suffering from glomerulonephritis, pyelonephritis, and various other renal anomalies. In these cases, the kidney has lost its ability to concentrate the urine because of tubular damage.
An abnormally high urine specific gravity also is indicative of a diseased state. For example, the urine specific gravity is abnormally high in an individual suffering from diabetes mellitus, adrenal insufficiency, hepatic disease or congestive cardiac failure. Urine specific gravity likewise is elevated when an individual has lost an excessive amount of water, such as with sweating, fever, vomiting and diarrhea. In addition, abnormally high amounts of nonionic urinary constituents, like glucose and protein, increase the urine specific gravity to 1.050 or greater in some individuals suffering from diabetes mellitus or nephrosis. Urine with a fixed low specific gravity of approximately 1.010 that varies little from specimen to specimen is known as isothenuric. This condition is indicative of severe renal damage with disturbance of both the concentrating and diluting abilities of the kidney.
In order to determine if an individual has either an abnormally high or an abnormally low urine specific gravity, and in order to help monitor the course of a medical treatment to determine its effectiveness, simple, accurate and inexpensive specific gravity assays have been developed. In general, the specific gravity of a test sample is a measurement that relates to the density of the test sample. The specific gravity is a value derived from the ratio of the weight of a given volume of a test sample, such as urine, to the weight of the same volume of water under standardized conditions (Eq. 1). ##EQU1## Water has a specific gravity of 1.000. Since urine is a solution of minerals, salts, and organic compounds in water, the specific gravity of urine is greater than 1.000. The relative difference reflects the degree of concentration of the urine specimen and is a measure of the total solids in urine.
Several methods are available to determine the specific gravity of urine. The most widely used method, and possibly the lease accurate, employs a urinometer. The urinometer is a weighted, bulb-shaped instrument having a cylindrical stem containing a scale calibrated in specific gravity readings. The urinometer is floated in a cylinder containing the urine sample, and the specific gravity of the urine is determined by the depth the urinometer sinks in the urine sample. The specific gravity value is read directly from the urinometer scale at the junction of the urine with the air. The urinometer method is cumbersome and suffers from the disadvantages of: a) requiring large volumes of urine test sample, b) a difficult and inaccurate reading of the urinometer scale, and c) unreliable assays because the urinometer is not regularly recalibrated.
Refractometry provides an indirect method of measuring the specific gravity of urine. The refractive index of urine is directly related to the number of dissolved particles in urine and, therefore, is directly related to the specific gravity of urine. Consequently, measurement of the refractive index of urine can be correlated to the specific gravity of urine. The refractometer method of determining urine specific gravity is desirable because specific gravity measurements are performed on as little as one drop of urine. However, the refractometer has the disadvantages of requiring daily calibration and not being amenable to home assays.
The falling drop method is another method of assaying for specific gravity which, like the urinometer, directly measures urine specific gravity. In this method, a drop of urine is introduced into each of a series of columns filled with solvent mixtures of increasing and known specific gravity. When the drop of urine comes to rest after its initial momentum has dissipated, and then neither rises nor falls, the specific gravity of the urine is determined to be identical to the specific gravity of the solvent mixture of that particular column. The falling drop method, however, is not widely used in routine urinalysis because of the lengthy time requirements in setting up such a assay and the inability of an individual to perform the assay at home.
The falling drop method described above also can be performed instrumentally. The instrument-based assay uses a specially designed column filled with a silicone oil having a controlled specific gravity and viscosity. The column is designed to measure the time required for a precisely measured drop of test sample to fall a distance defined by two optical gates (lamp-phototransistor pairs) mounted one above the other in a temperature-controlled column filled with a water-immiscible silicone oil of a slightly lower density than the test sample. The falling time is measured electronically and computed into specific gravity units. This specific gravity method is very precise, but the cost of the assay instrument and the degree of skill required to operate the instrument makes home testing for urine specific gravity impractical.
Not one of the above-described specific gravity assay methods is suited to performing specific gravity assays outside a medical office or laboratory. Consequently, reagent impregnated test strips were developed to enable an individual to perform specific gravity assays at home. In general, the test strip assay developed for specific gravity determinations is an indirect assay method, wherein the test strip changes color in response to the ionic strength of the urine sample. The ionic strength of a test sample is a measure of the type and amount of ions present in a test sample. The specific gravity of a test sample is proportional to test sample ionic strength. Therefore, by assaying for the ionic strength of a test sample, the specific gravity is determined indirectly and semiquantitatively by correlating the ionic strength of the test sample to the specific gravity of the test sample.
The present day specific gravity nest strips are sample pH dependent, and comprise a carrier matrix impregnated with a reagent composition including a polyelectrolyte, such as a partially neutralized poly(methyl vinyl ether/maleic acid); a chromogenic indicator, such as bromothymol blue; and suitable buffering agents. The reagent composition is sensitive to the number of ions, or electrolytes, in the test sample, such that the polyelectrolyte of the reagent composition undergoes an ion exchange, and releases hydrogen ions to the test sample in exchange for cations present in the test sample in an amount relative to take ionic strength of the urine sample.
Therefore, as the concentration of electrolytes in urine increases (high specific gravity), more cations are available to exchange with the hydrogen ions present on the polyelectrolyte of the reagent composition. The overall result is a release of hydrogen ions into the urine sample, and a resulting pH decrease of the urine sample that causes a color transition of the bromothymol blue chromogenic indicator from blue-green to green to yellow-green in response to increased specific gravity. The resulting color transition, indicating a pH change of the solution caused by increasing ionic strength, i.e., increasing specific gravity, is empirically and semiquantitatively related to the specific gravity of the urine sample.
For test strips utilizing the partially neutralized poly(methyl vinyl ether/maleic acid) polyelectrolyte and bromothymol blue indicator, assays for specific gravity are performed on aqueous test samples having a specific gravity of about 1.000 to about 1.030. A reading of 1.000, or a blue-green color, indicates that the urine has a very low specific gravity, as demonstrated by the lack of a color transition of the chromogenic indicator dye. A specific gravity reading of about 1.005 to about 1.030 is signified by color transitions, from blue-green through green to yellow-green, that serve as reliable indicators of increasing specific gravity.
It would be extremely advantageous to have a simple, trustworthy method of quantitatively assaying for urine specific gravity that allows visual differentiation of specific gravity values of about 1.000 to about 1.050. By providing a quantitative method of determining urine specific gravity in an easy to use form, such as a dip-and-read test strip, the urine assay can be performed by laboratory personnel to afford immediate test results. The specific gravity assay results can be interpreted in conjunction with assays for other urine constituents, such that a diagnosis can be made without having to wait for assay results and medical treatment can be commenced immediately. Furthermore, the test strip method can be performed by an individual at home to estimate the specific gravity of the urine and therefore to help monitor the success of the medical treatment the individual is undergoing.
As will be described more fully hereinafter, the method of the present invention is independent of test sample pH, and allows the fast and trustworthy assay for ionic strength or specific gravity of urine and other aqueous test samples by utilizing a test strip having a test pad that incorporates a reagent composition comprising: (1) a strong polyelectrolyte and (2) an indicator that is capable of binding with the polyelectrolyte and undergoing a spectral shift (i.e., is met&chromatic) and is sensitive to pH changes. The reagent composition is buffered at a pH of about 3 or less. For low to medium specific gravity test samples (i.e., less than about 1.015), the reagent composition undergoes a color transition in response only to the ionic strength, or ion concentration, of the test sample. The color transition is directly related to the ionic strength of the test sample. For high specific gravity test samples (i.e., about 1.015 or greater), the reagent composition undergoes a color transition in response to both the ionic strength and the buffer capacity of the test sample. As will be demonstrated more fully hereinafter, the reagent composition provides sufficient assay sensitivity to allow the quantitative determination of ionic strength and specific gravity of low through high specific gravity test samples.
Any method of assaying for the ionic strength or the specific gravity of urine or other aqueous test samples must yield trustworthy and reproducible results by utilizing a reagent composition that undergoes a color transition in response to the ionic strength and buffer capacity, or to the specific gravity, of the test sample, and not as a result of a competing chemical or physical interaction, such as a preferential interaction with another test sample component, like protein or glucose. Additionally, the method and composition utilized in the ionic strength or specific gravity assay should not adversely affect or interfere with the other test reagent pads that are present on multiple test pad strips.
In accordance with the present invention, the reagent composition incorporated into the carrier matrix provides sufficient sensitivity and color differentiation to assay for ionic strength, and therefore assay for specific gravity. The method is useful for measuring test sample specific gravity from about 1.000 to about 1.040. In addition, although dry phase nest strips have been used to assay for specific gravity, no dry phase test strip has incorporated a strong polyelectrolyte and an indicator that is capable of binding to the polyelectrolyte and that is sensitive to pH changes, buffered at a pH of about 3 or less, in an assay method for test sample ionic strength or specific gravity that is essentially independent of test sample pH. In addition, the assay method is intentionally designed to be essentially independent of the buffering capacity of a low to medium specific gravity test sample, but is sensitive to the buffering capacity of high specific gravity test samples, thereby improving the correlation of ionic strength to specific gravity and providing a more accurate assay for specific gravity.
Prior patents disclose the polyelectrolyte-dye ion exchange chemistry utilized in the present-day specific gravity assay of urine. For example, Falb et al. U.S. Pat. No. 4,318,709 and Stiso et al. U.S. Pat. No. 4,376,827 disclose a polyelectrolyte-dye technique used to assay for urine specific gravity. Each patent teaches utilizing polyelectrolyte-dye chemistry to determine the specific gravity of urine by monitoring the color transition of the dye.
The Falb et al. and Stiso et al. patents each disclose a composition and a method wherein the cations present in the test sample induce an ion exchange with the polyelectrolyte, thereby introducing hydrogen ions into the test sample. The change in hydrogen ion concentration, i.e., pH, is detected by a pH indicator. Accordingly, the previously disclosed methods are sensitive to the pH of the aqueous solution, and no direct interaction between the indicator dye and the polyelectrolyte occurs.
In addition, test sample buffer capacity interferes with the methods of Stiso et al. and Falb et al. because the polyelectrolyte-dye is buffered at a pH of about 6 to about 8 (which is within the urine pH range of about 5 to about 9). Test sample buffer capacity counteracts the introduction of hydrogen ions into the test sample by the polyelectrolyte, adversely affects the color transition, and thereby reduces the accuracy of the assay.
As used here and hereinafter, the term "metachromatic dye" is defined as a dye capable of undergoing a spectral shift upon binding to a polyelectrolyte, as opposed to a color transition due to a pH change. Accordingly, the term "metachromatic dye" encompasses: (1) dyes conventionally termed metachromatic which do not respond to changes in pH, i.e., "pH-insensitive metachromatic dyes" and (2) pH indicator dyes that bind to polyelectrolyte and undergo a color transition at a pH about 1 to 2 units below the pK.sub.a of the dye as a result of a polyelectrolyte-dye interaction as opposed to a pH change, i.e., "pH-sensitive metachromatic dyes". Such dyes are capable of exhibiting a color change due to metachromasia and a pH change.
The composition and method of the present invention differ from the Stiso et al. and Falb et al. patents in that an indicator, like a metachromatic dye, first binds to the polyelectrolyte. The metachromatic dye also can be sensitive to pH changes and undergo a color transition and thereby act as a pH indicator. Alternatively, if the metachromatic dye is not sensitive to pH changes, a separate pH indicator dye can be included in the composition. In addition, the composition is buffered at a pH of about 3 or less, and, preferably the strong polyelectrolyte is present in the acid form.
Upon contact between the reagent composition and a test sample that includes metal cations, such as urine, the metal cations compete for available binding sites on the polyelectrolyte and displace a number of the metachromatic dye molecules from the polyelectrolyte. As will be discussed in more detail hereinafter, upon release from the polyelectrolyte, the spectral properties of the metachromatic dye molecules change and a color transition results. The color transition is directly proportional to the amount of metachromatic dye released from the polyelectrolyte, which in turn is directly related to the ionic strength of the test sample. The color change can be correlated, quantitatively, to the ionic strength of the test sample; and the ionic strength of the test sample can be correlated, quantitatively, to the specific gravity of the test sample.
In addition, for test samples having a high specific gravity (e.g., about 1.015 or greater), the primary color transition attributed to test sample ionic strength is augmented by the effects of test sample buffer capacity, which cause a secondary color transition in the released metachromatic dye or the pH indicator dye. Accordingly, and in contrast to the Falb et al. and Stiso et al. disclosures, the present method is independent of test sample pH because the primary color transition results from a pH-independent displacement of the metachromatic indicator dye from a polyelectrolyte, like the acid form of a poly(vinyl sulfate). For test samples having a specific gravity of about 1.015 or greater, the accuracy of the method is improved by the secondary color transition attributed to a pH change resulting from the buffer capacity of the test sample.
In accordance with an important feature of the present invention, the indicator utilized in the method and composition of the present invention is capable of binding to the polyelectrolyte, and is able to respond to the buffer capacity of a test sample having a specific gravity of about 1.015 or greater, when the composition is buffered at a pH of about 3 or less. Accordingly, a single dye that exhibits both properties, or a combination of dyes, can be used as the indicator in the composition. Buffering the composition at a pH of about 3 or less ensures that displacement from the polyelectrolyte and a pH change due to test sample buffer capacity in a high specific gravity test sample will have similar or synergistic effects on the color transition of the indicator. This is contrary to the methods of Stiso et al. and Falb et al.
The present invention provides a composition and method for the accurate determination of ionic strength and specific gravity of urine and other aqueous test samples by utilizing an indicator that is sensitive to test sample ionic strength and buffer capacity, wherein the reagent composition is buffered at a pH of about 3 or less. European Patent Application 0 349 934 discloses a test strip and method of determining specific gravity or ionic strength of a sample utilizing a composition including a buffer, a complex former and a pH indicator dye. The complex former can be a crown ether, a cryptand, a podand or a multifunctional liquid. The method disclosed in the European Application is pH dependent, and utilizes a standard pH indicator dye, such as bromothymol blue or thymol blue. European Patent Application 0 349 934 does not teach or suggest a metachromatic dye or a polyelectrolyte utilized in the present invention.
Greyson et al. in U.S. Pat. No. 4,015,462 discloses a support matrix incorporating osmotically-friable microcapsules containing a fluid including a dye. A portion of the microcapsules bursts upon contact with a test sample of low osmolality. A resulting release of the dye-containing fluid causes a color transition that is correlated to the specific gravity. However, the difficult production of the microencapsulated-containing supporting matrix is a serious disadvantage of the Greyson et al. method.
In contrast to the prior art, and in contrast to the presently available commercial test strips, the method of the present invention provides a sensitive measurement of test sample ionic strength and specific gravity by utilizing a reagent composition including an indicator capable of responding to test sample ionic strength and buffer capacity, such as 2-[4-dimethylamino)styrl]-1-methylpyridinium iodide or quinaldine red, and a strong polyelectrolyte, like a poly(vinyl sulfate) or a poly(styrenesulfonate), in the acid form and buffered at a pH of about 3 or less, wherein the method is essentially independent of test sample pH. The present reagent composition undergoes a sufficient color transition upon contact with a test sample to provide an accurate ionic strength or specific gravity assay. The accuracy of assays for high specific gravity test samples is improved by a supplementary color transition attributed to test sample buffer capacity. Hence, new and unexpected results are achieved in the dry phase reagent strip assay of urine and other aqueous test samples for ionic strength or specific gravity.